ANALYSIS  OF  THE  FRUIT  OF  RHAMNUS  FRANGULA 


BY 

J.  LOWE  HALL 

B.  S.  University  of  Illinois,  1919 


THESIS 

Submitted  in  Partial  Fulfillment  of  the  Requirements  for  the 

Degree  of 

MASTER  OF  SCIENCE 

IN  CHEMISTRY 

IN 

THE  GRADUATE  SCHOOL 

OF  THE 

UNIVERSITY  OF  ILLINOIS 


1921 


- 


■ 


CONTENTS . 


Page 


Acknowledgment 

Introductory  and  Historical  - - - - 
The  Material  ----------- 

Method  of  Procedure  -------- 

A.  The  Water-Insoluble  Fraction 
3.  The  Water-Soluble  Fraction  - 
C.  The  Seeds  --------- 

Discussion  of  Results 

Summary  -------------- 

Bibliography  ----------- 


i 

1 

8 

9 

10 


14 


16 


oo 

Cj  O 


24 

25 


ACKNO  WL  E DG  M EN r 


The  writer  takes  sincere  pleasure  in  expressing  thanks 
and  appreciation  to  Dr.  G . D.  Beal,  under  whose  able  help  and 
direction  this  work  has  "been  carried  out. 


Digitized  by  the  Internet  Archive 

in  2016 


https://archive.org/details/analysisoffruitoOOhall 


1. 


ANALYSIS  OP  THE  FRUIT  OP  RHAMNUS  FRANGULA. 


Introductory  and  Historical. 

While  this  work  has  to  do  only  with  the  fruit  of  Rhamnus 
Frangula,  no  consideration  of  the  subject  could  he  complete  with- 
out some  mention  of  the  other  parts  of  the  plant  as  well  as  some 
of  the  more  closely  related  snecies.  For  the  use  of  the  various 
parts  of  these  plants  in  medicine  has  a very  ancient  origin,  and 
has  been  of  prime  importance  in  its  chemical  history  and  econom- 
ic application.  The  writer  is  indebted  to  an  article  written  by 
E.  N.  Gathercoal  (1)  in  the  Journal  of  the  American  Pharmaceuti- 
cal Association  from  which  the  following  brief  historical  con- 
sideration is  taken. 

For  centuries  the  bark  of  a wild  shrub,  known  in  England  as 
Alder  Buckthorn  or  Berry  Alder,  has  been  used  in  Eurone  as  a 
purgative.  This  bark  is  now  recognized  in  most  of  the  leading 
pharmacopoeias  of  the  world,  under  the  name  of  Frangula,  or 
Frangulae  Cortex.  Rhamnus  Frangula,  the  plant  yielding  the 
drug,  ranges  along  roadsides  and  in  thickets  over  all  of  Europe, 
except  in  the  very  northernmost  parts,  and  east  over  northern 
Asia . 

Associated  with  Rhamnus  Frangula  is  Rhamnus  Catharticus,  a 
thorny  shrub,  named  in  England,  Buckthorn  or  Way  thorn.  This 
plant  is  also  found  in  northern  Africa,  India,  and  eastern 


2 


United  States.  The  fruit,  especially,  has  been  employed  for  many 
centuries  in  Europe  as  a cathartic.  It  is  now  official  in  a few 
of  the  Euronean  pharmocopoeias . As  a medicine  the  fresh,  rice 
berries  are  made  into  a decoction  or  the  abundant  juice  is  ex- 
pressed and  made  into  a syrup.  The  bark,  also,  possesses  purga- 
tive properties,  which,  in  the  fresh  bark,  are  said  to  be  more 
drastic  than  Frangula. 

Another  group  of  Rhamni  furnishin^nedicinal  barks,  is  found 
along  the  western  coast  of  North  America.  -With  the  settlement  of 
California  by  the  Spaniards,  the  new-comers  noted  that  the  native 
Indians  used  the  bark  of  a certain  kind  of  a shrubby  tree  as  a 
cathartic.  The  Spaniards  named  this  plant  and  its  bark  Cascara 
Sagrada  (Sacred  Bark).  This  drug  is  now  official  in  nearly  all 
the  pharmacopoeias  of  the  world.  It  is  obtained  from  the  Plant 
Rhamnus  Purshiana,  which  ranges  over  the  west  slopes  of  the 
Cascade  Mountains,  from  central  California  well  up  into  British 
Columbia,  and  forms  extensive  low  forests  on  the  valley  and 
mountain  sides. 

The  term  "ramnos"  used  by  the  early  Greek  physicians  and 
naturalists,  is  thought  to  be  derived  from  the  Celtic  "ram", 
signifying  a tuft  of  thorns  or  branches.  The  name  was  applied 
to  certain  thorny  plants  by  these  writers,  but  from  their  meager 
or  inaccurate  descriptions  it  is  impossible  to  establish  that 
the  plants  mentioned  by  them  were  any  of  the  Rhamni  as  we  know 
them  today . 

The  early  Anglo-Saxons  were  acquainted  with  purgative 
properties  of,  at  least,  the  Rhamnus  Catharticus,  for  we  find 


3. 


the  plant  mentioned  in  their  medical  writings  before  the  Norman 
Conquest.  The  juice  of  Way thorn  berries  is  described  as  an 
aperient  by  Welsh  physicians  at  the  beginning  of  the  thirteenth 
century . 

Crescentius  (1305)  mentions  Rhamnus  Catharticus  under  the 
name  Spina  Gervinae  and  describes  Rhamnus  Frangula  under  the  name 
Avornus,  mentioning  the  use  of  the  middle  bark  as  an  evacuant. 

It  is  not  until  Matthiolus  (2)  (1543)  in  his  commentary  on 
the  materia  medica  of  Dioscorides,  that  a good  description  of 
Rhamnus  Frangula,  with  mention  of  the  purgative  property  of  bark 
and  berries,  is  found  in  literature.  He  first  uses  the  name 
"frangula”  in  connection  with  the  plant;  (frango,  frangere, 
meaning  "to  break",  an  allusion  to  the  soft  and  fragile  nature 
of  its  wood). 

By  1700  the  botanical  characters  of  most  of  the  European 
Rhamni  were  well  established.  Linnaeus  (3)  (1753)  places  them 
in  the  Petandria  Monogynia.  He  includes  both  Rhamnus  Frangula 
and  Catharticus  as  natives  of  Sweden  in  his  Flora  Svecica  (1745), 
and  mentions  as  pharmaceutical  products  derived  from  them:  Spina 
Gervinae  Baccae,  Syrupus  Domesticus,  Frangulae  Cortex. 

The  bark  of  Rhamnus  Frangula  has  been  recognized  in  the 
pharmacopoeias  of  central  Europe  since  the  middle  of  the  last 
century,  including  the  Banish  (1368),  Norwegian  (1870),  Swedish 
(1871),  German  (1870),  Prussian  (1862),  Hanoverian  (1861),  and 
Dutch  (1871)*,  Austrian  (1389),  French  Codex  (1908),  U.  S.  (1880), 
and  Eritish  (1885,  though  it  was  omitted  from  the  last  edition). 

The  chemistry  of  rhamnus  barks  presents  much  of  interest 


4. 


because  from  the  first  analysis  "by  Gerber  in  1828,  it  has  been 
observed  that  the  active  urinciples  are  resinous  in  nature, 
difficult  to  separate  from  one  another  and  to  determine  their 
true  consti tution . Even  at  the  present  day  these  analyses  are 
far  from  being  in  a satisfactory  condition. 

Gerber,  (4)  obtained,  among  numerous  other  vegetable  con- 
stituents, 2.7$  of  yellow  resinous  coloring  matter  and  4.6$  of 
bitter  acrid  extractive,  which  he  considered  contained  the  active 
constituents.  He  noted  the  yellow  coloring  matter  became  dark- 
red  with  alkalies. 

Hubert  (5)  (18.30)  analyzed  the  juice  from  the  fruit  of 
Rhamnus  Catharticus.  He  found  a bitter  substance,  apparently 
the  active  constituent,  and  closely  resembling  the  cathartin 
of  senna  leaves,  a green  coloring  matter,  which  in  the  ripe 
fruit  is  purple  red,  due  to  the  action  of  acids  in  the  ripening 
fruit,  and  a brown  material  insoluble  in  alcohol  but  easily  sol- 
uble in  water. 

Pleury  (6)  (1842)  obtained  from  the  unripe  berries  of 
Rhamnus  Catharticus,  rhamnine  in  pale  yellow  crystals. 

Winckler  (7)  (1849)  obtained  rhamnine  from  the  unripe 
berries  of  Rhamnus  Catharticus  and  cathartin  from  the  ripe 
fruit.  He  considered  that  rhamnine  by  the  ripening  process  is 
converted  into  cathartin  and  glucose.  (This  is  the  first  pub- 
lished evidence  of  the  glucosidic  nature  of  these  resinous  con- 
stituents of  the  Rhamni ) . 


5 . 

Binswanger  (8)  (1849)  found  in  frangula  bark  the  crystalliz- 
able  yellow  coloring  principle  which  was  named  (by  L.  A.  Buchner) 
rhamnoxanthin , an  ether-soluble  amorphous  resin,  one  or  more 
alcohol  soluble  resins,  a bitter  substance  of  resinous  nature 
in  which  the  ourgative  nrooerties  of  the  bark  seem  to  lie,  sugar, 
gum,  tannin,  plant  acids,  extractive,  etc.  He  compared  the 
bark  of  Rhamnus  Catharticus  with  Prangula  bark  and  found  that 
the  constituents  were  similar,  but  included  also  a bitter, 
water  soluble,  crystallizable  substance  to  which  he  attributed 
the  greater  hydragogue  properties  of  the  Rhamnus  Catharticus 
bark.  This  principle  was  differentiated  from  the  cathartin  of 
senna  leaves  and  named  rhamno-cathartin . He  found  rhamno- 
xanthin also  in  the  seeds  of  Rhamnus  Catharticus  and  Rhamnus 
Prangula.  The  juice  of  the  rioe  berries  contained  a violet 
coloring  matter  turning  red  with  acids  and  green  with  alkalies, 
a bitter  extractive,  etc.  The  unripe  berries  contained  only 
the  rhamnin  of  Pleury. 

Buchner  (9)  (1853),  who  worked  with  Binswanger  at  Munich 
in  1849,  obtained  from  the  root  bark  of  Rhamnus  Prangula, 
rhamnoxanthin  in  sublimable,  golden-yellow  needles,  very 
slightly  soluble  in  water,  but  easily  so  in  alcohol  or  ether 
(especially  hot),  readily  in  solutions  of  ammonia  and  the 
fixed  alkalies  with  a fine  -nurole-red  color,  and  in  concentrated 
sulfuric  acid  with  a red  color.  By  neutralization  of  the  al- 
kaline solution,  the  rhamnoxanthin  was  thrown  out  as  a yellow 
powder,  and  by  dilution  of  the  concentrated  sulfuric  acid  so- 
lution with  water  it  was  likewise  separated  out. 


6 . 

Casselmann  (10)  (1857)  obtained  the  resinous  constituent 
of  Prangula  in  crystalline  form,  designated  it  frangulin,  and 
decomposed  it  with  the  formation  of  glucose  and  an  acid  product 
he  named  frangulinic  or  ni tro-frangulic  acid. 

Phipson  (11)  (1858)  found  rhamnoxanthin  in  the  branches  of 
Rhamnus  Frangula  and  of  Rhamnus  Catharticus  and  corroborated 
Buchner's  description  of  it. 

Kubly  (12)  (1866)  separated  from  frangula  bark  the  glucos- 
ide,  which  he  named  avornin,  an  amornhous  resin,  and  a principle 
similar  to  cathartic  acid,  which  he  had  a short  time  previously 
isolated  from  senna  leaves.  The  avornin  he  split  into  avornic 
acid  and  glucose. 

Paust  (13)  (1869)  stated  that  the  frangulin  of  Casselmann, 
and  the  avornin  of  Kubly  are  identical,  and  assigned  them  the 
formula  C^H^O^.  He  named  the  acid  resin  from  the  decompo- 
sition of  this  glucoside,  frangulic  acid. 

Liebermann  and  Waldstein  (14)  (1876)  identified  emodin 
( trioxymethylanthraquinone ) from  frangula  bark  and  stated  that 
frangulic  acid  is  probably  emodin. 

Prescott  (15)  (1879)  was  the  first  to  analyze  cascara  sa- 
grada  bark.  He  found  a brown  resin  of  strongly  bitter  taste, 
colored  a vivid  purple-red  by  potassium  hydrate  solution, 
sparingly  soluble  in  water  or  ether,  but  freely  so  in  alcohol, 
chloroform,  benzol,  carbon  disulphide,  and  solutions  of  caustic 
alkalies,  though  precipitated  from  the  latter  by  acids.  He 
found  also  some  other  resins,  tannin,  oxalic  and  malic  acids. 


etc . 


7. 


Schwab e (16)  (1888)  found  frangula  to  yield  frangulin  0.04$, 
and  eraodin  0.1$.  He  corroborated,  the  physical  characters  of 
frangulin  as  stated  by  Casselman  and  Faust,  and  amplified  on 
them.  His  -proximate  analysis  indicated  the  formula 
Frangulin  by  hydrolysis,  yields  emodin.  He  round  in  case <■. r a bark 
emodin  but  no  frangulin. 

Thorpe  and  Miller  (17)  (1892)  corroborated  Schwabe's  form- 
ula for  frangulin  and  determined  that  the  sugar  from  the  deco  — 
position  of  frangulin  was  a true  rhamnose. 

Cabannes  (18)  (1895)  renorted  that  in  sections  of  frangula 
bark  treated  with  alcoholic  potassa  solution,  the  parenchyma  of 
the  cortex,  medullary  rays,  and  bast,  all  acquire  a strong  red 
color,  but  that  in  cascara  bark  sections  only  one  or  two  layers 
of  the  cortical  parenchyma,  the  medullary  rays,  and  the  five  or 
six  inner  rows  of  bast  parenchyma  take  the  color.  Ammonia  and 
soda  solutions  react  the  same  as  potassa. 

Oesterle  (19)  (1399)  found  that  frangula-emodin  differs 
from  aloe-emodin. 

Perrot  (20)  (1900)  stated  that  powdered  frangula  bark  with 
alkalies  produces  a deen  red  color,  but  that  powdered  cascara 
bark  gives  a yellow  color,  and  that  the  powders  could  be  dis- 
tinguished in  this  manner. 

Tschirch  and  Polacco  (21)  (1900)  analyzed  Rhamnus  Catharti- 
cus  fruit  and  determined  the  presence  of  emodin,  several  color- 
ing matters,  a sugar,  etc.  The  purgative  action  was  ascribed 


to  the  emodin. 


3 . 

Tschirch  and  Pool  (22)  (1908)  found  that  the  emodins  from 
frangula  and  cascara  "barks  were  identical,  that  neither  of  the 
barks  yielded  rhein,  but  that  chrysophanic  acid  was  present  in 
frangula  bark. 

Schmidt  (23)  (1912)  describes  frangulin  (rhamnoxanthin ) , 

(C  H 0 ),  as  occuring  in  lemon-yellow,  glistening  fine  needle- 

Cj  X oU  »/ 

crystals,  odorless  and  tasteless,  melting  at  228°  to  230°  C.  It 
is  almost  insoluble  in  water  and  in  cold  ether,  but  soluble  in 
180  parts  of  80^  hot  alcohol.  Concentrated  sulfuric  acid  dis- 
solves it  with  a dark-red  color  and  with  caustic  alkalies  it 
forms  solutions  of  a ourple  red  color.  By  boiling  with  an  al- 
coholic solution  of  hydrochloric  acid  it  becomes  converted  into 
rhamnose  and  frangula-emodin , (C-^H^O^ ) , which  forms  bright  red 
glistening  needles  melting  at  255°C.  It  is  insoluble  in  water, 
slightly  soluble  in  alcohol,  and  easily  soluble  in  chloroform 
and  benzol . 


The  Material. 


The  berries  of  Rhamnus  Frangula  were  gathered  as  they 
became  ripe  and  immediately  immersed  in  95  per  cent  alcohol, 
thus  preventing  any  enzymatic  action.  About  a half  liter  of 
the  berries  covered  with  alcohol  was  available  for  this  analysis 
Consequently  the  work  was  handicanned  considerably  in  attempting 
to  purify  and  identify  small  amounts  of  material. 


. 


’ A O 

* 


r i .-tv  , - ■ — r i,, . 

9. 

Method  of  Proc edure . 

The  alcoholic  extract  was  filtered  from  the  berries  and 
the  berries  washed  with  about  200  c.c.  of  alcohol  (till  the 
filtrate  was  colorless).  The  extract  was  browni sh -black  by- 
reflected  light,  and  when  quite  shallow  transmitted  a dull, 
dark-red  light. 

The  berries  were  then  dried  in  a vacuum  oven  at  70c'~  80°C, 
and  mulled  in  a mortar  in  order  to  breav  the  hardened  skins  from 
the  seeds.  The  skins  were  winnowed  from  the  seeds  and  exhausted 
with  boiling  alcohol,  which  was  then  combined  with  the  first 
extract . 

This  combined  alcoholic  extract  was  evaoorated  to  a thick 
syrup  consistency,  and  to  it  was  added  300-400  c.c.  of  water. 
This  mixture  was  placed  in  a refrigerator  for  preservation 
while  the  water-soluble  material  was  going  into  solution.  The 
gelatinous  precipitate  was  filtered  out,  washed  with  water,  and 
again  taken  un  in  alcohol,  although  an  insoluble  residue  re- 
mained, as  was  often  the  case  in  similar  subsequent  operations; 
itj^ay  have  been  the  result  of  resinif ication,  hydrolysis,  or 
similar  reaction.  Sufficient  alcohol  was  added  to  the  aqueous 
solution  for  preservation . 

The  dry  seeds  (to  be  analyzed  later)  were  put  in  a dry 


stoppered  flask 


10. 


A. The  Water-Insoluble  Fraction. 

The  accompanying  diagram  illustrates  the  procedure  followed 
in  manipulating  the  alcoholic  extract,  the  Roman  numerals  to  he 
used  later  in  referring  to  the  individual  oortions  in  their  sub- 
sequent separations. 

The  water-insoluble  portion  of  the  original  extract  having 
been  taken  up  in  alcohol  as  previously  mentioned,  was  then 
mixed  with  a sufficient  quantity  of  purified  sawdust  to  absorb 
it  effectively,  allowed  to  dry  until  the  odor  of  alcohol  was  no 
longer  apparent,  and  then  placed  in  a large  Soxhlet  extraction 
apparatus.  The  material  was  then  extracted  with  ether  until 
it  came  through  colorless  (II,  see  diagram).  Extraction  was 
then  completed  with  alcohol  (V,  see  diagram). 

The  ether  extract  (II)  gave  a strong  color  test  (red)  with 
ammonia,  and  was  therefore  shaken  out  successively  with  5 per 
cent  solutions  of  ammonium  carbonate,  sodium  carbonate,  and 
sodium  hydroxide;  these  fractions  were  acidified  and  shaken 


out  with  ether. 

Vol . of  base 

Color 

Comnarat 

i ve 

Fraction 

required 

precipi tation 

Ammonium  carbonate 

75  c . c . 

Faint  pink 

Hone 

Sodium  carbonate 

230  c.c. 

Very  deep  red 

O 

c» 

Sodium  hydroxide 

200  c.c. 

Pale  red 

1 

The  precipitates  from  the 

second  and  third 

fractions , 

( the 

first  was  discarded) 

, were  soluble  in  glacial  acetic  acid. 

but 

did  not  crystallize 

out  on  concentration,  a small  fraction 

merely  separating  in 

a semi -gelatinous  state. 

This  material 

- 


. 


: ' - 


; 


( 


* 


’ 


• ■ 


11. 


DIAGRAM  SHOWING  MANIPULATION  OP  COMBINED  ALCOHOLIC 

EXTRACT. 


Alcoholic  Extract 

■ r 

Precipitate  with  water 


1 


Insolubl e in  water 
Take  up  in  95^  alcohol 


1 


Soluble  in  water  ( V III ) 

Shake  out  with  ether 
and  combine  extract 
with  extraction  (II) . 


Sol,  in  alcohol  (I) 

Mix  with  purified 
sawdust  and  extract 
with  ether  in 
soxhlet  (II ) . 

Complete  extraction 
with  alcohol  (V). 

Hydrolyze  with  1^ 
HCL.  Concentrate  to 
syrup  and  pour  into 
water.  


Insol,  in  alcohol 
Resin;  discarded. 


J 


n 


r — 

Precipi tate 


Decolorize  aqueous 
solution  with  lead 
subacetate,  filter. 

~i 


i 


Recover  acids  by 
passing  H?S  into 
aqueous  suspension. 
Filter  off  PbS. 

Aqueous  solution  (III). 


Filtrate  (IV). 
Determine  sugars 


1 


Soluble  in  water  (VI ) Insoluble  in  wate'r 


Shake  out  with  ether  Dry  and  extract  with 

and  combine  washings  ether  in  soxhlet  (VII). 

with  (VII).  Examine 
for  carbohydrates. 

Test  small  amount  of  each  ether  extract  for_ 
anthraquinone  derivatives  with  a little  ammonia 
(deep  red  or  purple). 


12. 


in  each  case  was  filtered  off  on  a hard  filter  and  dried,  but 
amounted  -practically  to  nothing.  The  acetic  acid  solution  was 
a very  deep,  rich  red  color,  nearly  opaque.  The  addition  of 
water  to  each  of  these  acetic  acid  solutions  caused  the  pre- 
cipitation of  a bright  yellow  substance  in  a flocculent  state, 
which  was  filtered  out,  redissolved  in  5^  sodium  hydroxide, 
acidified,  and  shaken  out  with  ether.  On  allowing  the  spon- 
taneous evaporation  of  the  ether  solution  of  the  sodium  carbon- 
ate fraction  on  a large  watch  glass,  it  was  noticed  that  the 
material  aopeared  to  be  deposited  in  two  forms,  an  outer  ring 
of  a deep  red  leaf-like  material,  and  an  inner  circle  of  small 
yellow  particles,  - mostly  the  former. 

The  material  in  each  case  was  again  taken  up  in  glacial 
acetic  acid  and  concentrated  on  the  steam  bath.  During  this 
purification,  the  sodium  hydroxide  fraction  decreased  markedly 
in  mass,  and  the  acetic  acid  solution,  even  when  concentrated 
to  about  1 c.c.,  yielded  only  a few  particles  of  apparently 
flocculent,  non-crystalline  material. 

However  the  sodium  carbonate  fraction  under  similar  treat- 
ment yielded  a small  cron  of  very  finely  divided  material, 
melting  at  250°-  2^5°  C.  An  attempt  to  nrenare  an  acetyl 
derivative  yielded  barely  enough  material  of  questionable  nature 
for  a melting  point  determination:  it  did  not  melt  under 

320°0 . 

From  these  data  it  is  evidently  impossible  to  arrive  at 
any  definite  conclusions  concerning  the  identity  of  the  sub- 
stances present,  but  it  seems  probable  that  the  sodium  carbonate 


13. 


fraction,  at  least,  contained  e’nodin  (red;  melts  at  255°C),  and 
the  sodium  hydroxide  fraction  possibly  chrysophanic  acid 
(yellow,  though  melting  much  lower,  198°C,  and,  according  to 
Beal  and  Okey  (24),  insoluble  in  cold  solutions  of  alkali  car- 
bonates, but  soluble  in  sodium  hydroxide). 

The  alcoholic  extract  (V,  see  diagram)  was  hydrolyzed  3-l/2 
hours  on  the  steam  bath  with  100  c.c.  1 % hydrochloric  acid.  It 
was  then  evaporated  to  a syrup,  diluted  with  considerable  water, 
and  the  precipitate  filtered  off  and  dried.  The  water  solution 
was  pale  yellow.  The  dry  precipitate  wa s extracted  with  ether 
in  a^Soxhlet  apparatus.  The  aqueous  solution  (VI ) was  shaken 
out  with  ether,  the  extract  being  combined  with  the  Soxhlet 
extract  (VII ).  The  aqueous  solution  did  not  reduce  Fehling's 
solution.  The  Molisch  •i-naohthol  test  for  carbohydrate 
(Sherman  (25)  p.  57)  was  negative  in  comparison  to  a blank  on 
distilled  water.  Therefore,  as.  the  subsequent  examination  of 
the  ether  extract  (VII ) shov/ed  considerable  anthraquinone  de- 
rivative, it  must  have  -been  linked  in  some  form  insoluble  in 
ether,  but  not  with  a carbohydrate. 

The  ether  extract  (VII ) gave  a very  pronounced  color  test 
with  dilute  alkali.  Accordingly  It  was  extracted  with  the 
usual  sequence  of  dilute  bases. 

Fraction  Vol . of  base  Color  Comparative 

required Precipitation 

Ammonium  carbonate  150  c.c.  Pale  yellow  Trace 

Sodium  carbonate  450  c.c.  Very  deep  red  Very  heavy 

Sodium  hydroxide  100  c.c.  Pale  red  Trace 


14. 


The  sodium  carbonate  fraction  contained  perhaps  0.2  gram 
of  precipitated  material,  which  was  crystallized  from  hot  gla- 
cial acetic  acid,  yielding  a small  crop  of  minute  crystals, 

which  appeared  under  the  microscope  as  broad,  ragged  nlates, 

o o 

and  which  melted  at  252  - 255  G.  They  were  deem  red  in  color, 

hut  reflected  a yellow  fluorescent  color  similar  to  "fool’s 
gold".  The  substance  was  evidently  emodin.  The  other  two 
fractions  yielded  negligible  quantities  of  precipitate. 

B.  The  Water-Soluble  Fract .ion . 

The  aqueous  solution  (VIII)  was  shaken  out  with  ether,  but 
the  extract  gave  no  color  test  with  alkali.  The  ether,  was 
evaporated  on  a large  watch  glass,  leaving  a thin  film  of 
light-yel lowi sh  brown  material  possessing  a deasant  odor  re- 
sembling peaches,  and  a bitter  taste.  It  had  the  appearance 
of  a gum  and  did  not  reduce  Fehling’s  solution  before  or  after 
hydrolysis,  was  insoluble  in  water,  hot  ten  per  cent  sodium 
hydroxide,  and  acetic  anhydride. 

The  aqueous  solution  (VIII ) was  treated  with  lead  sub- 
acetate to  remove  its  deep  color,  the  precipitate  filtered  off, 
and  hydrogen  sulfide  passed  into  the  filtrate  to  remove  the 
excess  lead.  After  the  lead  sulfide  was  filtered  off,  the 
filtrate  was  colored  only  slightly  yellow.  This  solution  (IV) 
was  warmed  gently  on  the  steam  bath  and  air  bubbled  through  it 
to  remove  the  hydrogen  sulfide. 


15. 


The  Molisch  oc-naphthol  test  gave  decided  indication,  of 
carbohydrate  in  the  solution  even  to  the  extent  that  consider- 
able precipitate  was  formed.  The  solution  exerted  a very 
marked  reduction  on  Fehling's  solution,  and  formed  an  osazone 
in  8-9  minutes  which  under  the  microscope  appeared  as  the 
sheaves  of  long  yellow  needles  characteristic  of  glucosazone; 
it  melted  at  198-202°  C.  Consequently  the  sugar  might  be 
either  glucose  or  levulose.  However,  Seliwanoff’s  test  for 
levulose  (Hawk  (26)  p.  55)  was  negative;  therefore  there  must 
have  been  glucose  present  in  appreciable  quantity.  A very 
brilliant  red  anilin  acetate  test  for  furfural  was  obtained, 
indicating  a pentose  (Sherman  (25)  p.  57). 

No  positive  test  for  rhamnose  was  obtained  by  the  alcohol - 
sulfuric  acid  method  (Browne  (27)  o.  577). 

The  lead  subacetate  precipitate  was  pulverized,  suspended 
in  water,  and  hydrogen  sulfide  passed  in  until  the  lead  was 
converted  to  the  sulfide,  thereby  liberating  the  organic  acids 
carried  down  by  the  subacetate.  Tannic,  malic,  and  a trace  of 
succinic  acids  were  found  by  a system  of  analysis  according  to 
Barfoed  (28).  Briefly  Barfoed's  method  is  as  follows: 

Neutralize  the  solution  with  ammonia,  concentrate  to  small 
volume,  neutralizing  again  if  necessary.  Mix  with  7-3  volumes 
of  alcohol  and  allow  to  stand  12-24  hours;  filter.  The  pre- 
cipitate contains  oxalic,  tartaric,  and  citric  acids;  malic 
acid  will  appear  in  the  filtrate  and  may  be  precipitated  with 
lead  subacetate.  Tannic  acid  is  mostly  precipitated  immediately 


16. 


in  slightly  ammoniacal  solution  by  calcium  chloride.  Lead  sub- 
succinate  is  soluble  in  hot  water,  the  submalate  is  not.  Or 
treat  the  alkali  salts  of  succinic  and  malic  acid  with  lead  sub- 
acetate till  precipitation  is  complete,  add  ammonium  acetate 
until  precipitate  dissolves,  and  add  two  volumes  of  alcohol;  the 
lead  malate  precipitates,  and  the  succinate  remains  in  solution. 
Calcium  malate  is  insoluble  in  a 50-70  oer  cent  solution  of 
alcohol  (by  volume)  in  water.  Neutralized  benzoic,  acetic,  and 
formic  acids  do  not  precipitate  on  addition  of  calcium  chloride 
and  1-2  volumes  of  alcohol. 

C.  The  Seeds . _ 

The  seeds  are  dicotyledonous , the  cotyledons  being  flat 
and  round,  and  lying  parallel  to  the  flat  side  of  the  seed; 
ordinarily  two  seeds,  sometimes  three,  occur  in  each  berry  and 
are  flattened  against  each  other.  A thin  cross-section  of  a 
cotyledon  was  mounted  on  a slide  and  treated  with  5%  sodium 
hydroxide.  Under  the  microscope  a row  of  red  snots  appeared 
along  the  axis  of  the  mount,  which  followed  the  location  of  the 
vacuoles,  thus  demonstrating  the  distribution  of  the  anthra- 
quinone  derivatives  in  the  seed. 

The  dry  seeds  were  ground  up  in  a coffee  mill  but  with 
considerable  difficulty  caused  by  clogging;  their  oil  content 
was  such  as  to  allow  the  ground  seed  to  cohere  tightly  together. 
Thirty-two  grams  of  the  ground  seed  were  obtained;  it  possessed 
a oleasant/nut-like  odor,  and  was  of  a dark  chocolate  brown  color. 


17. 


This  material  was  extracted  in  a Soxhlet  apparatus  with 
ether.  The  extract  gave  a deep  red  coloration  with  dilute  alka- 
li, and  was  shaken  out  with  dilute  sodium  hydroxide,  in  order  to 
get  an  approximate  idea  of  the  total  anthraquinone-deri vative 
content.  The  alkali  extract  was  acidified  with  dilute  hydro- 
chloric acid,  the  yellow  nrecipitate  filtered  off,  carefully 
dried  and  weighed;  it  amounted  to  0.6  gram,  or  about  two  per 
cent  of  the  weight  of  the  dry  seeds.  This  material  would  not 
again  go  entirely  into  solution  in  ether,  chloroform,  or  mix- 
tures of  "both,  or  in  benzene.  This  conduct  was  noted  at  other 
times  when  the  material  was  allowed  to  dry  and  warmed  below 
100°  G.  Apparently  some  constituent  of  it  oxidizes  readily. 
However  the  material  seemed  most  soluble  in  benzene,  and  in 
such  solution  was  shaken  out  with  the  usual  succession  of  dilute 
bases . 


Fraction 


Vol . of  base 
required 


Comparative 

Color  of  Color  of  nre- 
Ex tract  Benzene _ cini tation 


Ammonium  carbonate  150  c.c. 
Sodium  carbonate  200  c.c. 
Sodium  hydroxide  225  c.c. 


Pink  Red  trace 

Rich  red  Yellow  1 

Dark  red  Colorless  2 


The  first  fraction  was  discarded.  The  second  fraction 
failed  to  crystallize  from  hot  benzene  or  alcohol,  merely  se- 
parating as  a small  amount  of  bright  yellow  amorphous  material. 

The  third  fraction  likewise  would  not  crystallize  from 
benzene  or  alcohol.  During  the  evaooration  of  the  alcohol  from 
this  fraction  on  the  steam  bath,  the  material,  on  being  exnosed 
to  the  air,  even  before  all  the  alcohol  was  gone,  turned  black 


. 


18. 


and  acquired  a sticky  consistency.  It  would  not  then  completely 
redissolve  in  alcohol,  a 'black  sediment  remaining.  The  solution 
was  decanted  from  the  sediment  and  allowed  to  evaporate  snontan- 
eously;  the  denosit  was  dark  purplish  brown.  This  experience 
serves  to  show  how  carefully  the  material  must  be  handled  to 
prevent  oxidation. 

The  remaining  ether  extract  of 'the  seeds  was  evaporated  on 
the  steam  bath,  leaving  a clear  mobile,  pale-yellow  oil  possess- 
ing the  pleasant  odor  characterizing  the  ground  seeds.  This  oil 
was  dried  in  vacuo  over  concentrated  sulfuric  acid,  and  the 
following  constants  determined: 

The  index  of  refraction  was  taken  by  an  Abbe  ref ractome ter 
at  19°  C.  and  corrected  to  15.5°  G. 

Reading  at  19°  C --------  1.4726 

Reading  corrected  to  15.5°  G - - 1.4749 

The  specific  gravity  was  determined  with  a Westphal  balance 
by  the  alcohol-water  method.  A few  drops  of  the  oil  were  nut 
in  a cold  mixture  of  alcohol  and  water,  and  the  mixture  adjusted 
by  adding  alcohol  or  water  until  the  droplets  of  oil  remained 
stationary  in  suspension  at  15.5°  G.  At  this  point  the  snecific 
gravity  of  the  mixture  was  read  on  the  Westphal  balance. 

Snecific  gravity  of  oil  at  15.5°  G - - - 0.9197 
The  iodine  number  was  determined  according  to  the  method  of 
Wijs.  The  iodine  monochloride  solution  in  glacial  acetic  acid 
was  added  to  the  sample  (0.2  - 0.4  gram)  dissolved  in  chloro- 
form, and  contained  in  a glass  stoppered  flask.  After  stand- 
ing thirty  minutes  a solution  of  potassium  iodide  was  added. 


■* 


19 


and  the  free  iodine  titrated  with  standard  sodium  thiosulphate 
solution.  A blank  de termination  was  made  at  the  same  time. 
Thiosulfate  solution,  1 c.c.  = 0.01376  gram  iodine. 


Weight  of  samnle 

I II 

0.2382  0.2932 

Thiosulfate  titration 


Blank 

47.00  c.c.  47.00  c.c. 

Samnle 

26.78  22.13 

Net 

20.22  24.82 

Iodine  absorbed 

0.2780  g.  0.3416  g. 

Iodine  number 

116.7  117.1 

The  saponifi cation  number  was  determined  by  refluxing  the 
sample  with  alcoholic  potash  on  a steam  bath  for  thirty  minutes, 
cooling,  adding  phenolphthalein,  and  titrating  with  standard 
acid.  A blank  determination  was  also  made. 

Normality  factor  of  acid  0.1003. 


Weight  of 'Sample 

I II 

2.1132  g.  2.0137  g. 

Acid  titration 


Blank 

97.31  c.c.  9J. 81  c.c. 

Samnle 

25.00  28.50 

Net 

72.31  69.31 

Saponification  number  193.9  193.7 

The  ner  cent  soluble  and  insoluble  acids  were  determined 
from  the  combined  solutions  from  the  sanonif ication  value 


de termination 


Two  cubic  centimeters  more  of  standard  acid 


' 


20. 


than  required  to  liberate  the  fatty  acids  from  the  soap  were 
added  to  the  solution.  The  mixture  was  slightly  warmed  to 
facillitate  cohesion  of  the  insoluble  acids,  and  transferred  to 
a separatory  funnel  whereby  the  floating  insoluble  acids  were 
senarated  and  washed.  The  acids  were  transferred  by  the  aid  of 
a little  ether  to  a small  weighing  bottle,  nlaced  in  a vacuum 
desiccator  over  concentrated  sulfuric  acid,  and  dried  to  constant 
weight . 


The  nercent  soluble  acids  was  determined  by  titrating  the 
aqueous  solution  obtained  above  with  standard  alkali,  allowing 
for  the  two  cubic  centimeters  excess  of  standard  acid  used. 


Weight  of  combined  samples 
Weight  of  insoluble  acids 


4.1269  g. 


3.3712  g. 


Per  cent  of  insoluble  acids  93.3 


Normality  factor  of  alkali 

Weight  of  combined  samnles  4.1269  g. 


0.0993. 


Titration  with  alkali 


4.90  c . c . 


Excess  standard  acid 


2.00 


Titration  of  soluble  acids  2.90 


Calculated  as  butyric 


0.02543  g. 


Per  cent  soluble  acids 


0.62 


The  index  of  refraction  of  the  insoluble  acids  was  taxen 
by  an  Abbe  ref ractometer , and  corrected  to  15.5°  C,  as  the  acids 
solidify  below  19°  C. 


Reading  ----- 
Corrected  to  15.5°  C 


1.4653 


1.4660 


1.4635 


1.4635 


21. 


The  melting  ooint  of  the  insoluble  acids  was  taken  by 
drawing  the  melted  acids  into  melting  point  tubes  about  one 
millimeter  in  diameter,  leaving  in  a refrigerator  24  hours, 
and  melting  in  the  usual  way  in  an  onen  beaker  of  water.  The 
melting  point  was  taken  as  the  point  at  which  the  acids  became 
transparent . 


Melting  point  of  insoluble  acids  - - - 26.5°  C. 

The  solidifying  point  of  the  acids,  or  titer  test,  was 
taken  by  surrounding  the  beaker  with  ice-water  and  noting  the 
point  at  which  turbidity  apoeared. 

o 

Solidifying  point  of  insoluble  acids  - - 19.5  0. 

The  iodine  number  of  the  insoluble  acids  was  determined 
in  the  same  way  as  for  the  oil. 

Thiosulfate  solution,  1 c.c.  = 0.01376  grams  iodine. 

I II 


Weight  of  samole 
Thiosulfate  titration 
Blank 
Sample 
Net 

Iodine  number 

Because  of  the  long  oeriod  of 


0.2176  g.  0.2246  g-. 

46.90  c.c.  46.90  c.c. 
50.65  29.90 

16.25  17.00 

101.9  101.6 

time  required  to  dry  the 
took  place,  since  the 
higher  than  that  of  the 

efficient  aspirator, 
carbon  dioxide 


insoluble  acids,  oxidation  evidently 
iodine  number  of  the  acids  should  be 
oil.  The  desiccator  was  evacuated  with  an 
and  the  nrecaution  of  drying  in  an  atmosnhere  of 
was  not  thought  necessary. 


22. 

The  neutral  equivalent  of  the  insoluble  acids  was  determin- 
ed by  dissolving  the  samples  in  neutral  alcohol,  adding  phenol- 
phthalein,  and  titrating  with  standard  alkali. 

Normality  factor  of  alkali  0.0998. 

I II 


Sample  0.5365  g.  0.4286  g. 

Titration  13.36  c.c.  14.65  c.c. 

Neutral  equivalent 

of  insoluble  acids  292.3  293.1 

The  oil  is  a characteristic  seed-oil,  resembling  in  these 
constants  sunflower-seed  oil,  and  cottonseed  oil,  but  having 
a slightly  lower  specific  gravity  and  less  viscous  behaviour. 


Discussion  of  Results. 

It  is  a notable  fact  that  the  sodium  carbonate  fraction 
of  the  alkali  extract  was  distinctly  the  largest  fraction  in 
the  case  of  the  alcoholic  extract  and  the  hydrolyzed  alcoholic 
extract,  but  in  the  case  of  the  seeds  the  sodium  hydroxide 
fraction  was  the  largest.  However,  the  solvent  in  the  first 
two  cases  was  ether,  whereas  benzene  was  used  in  the  latter, 
which  might  explain  the  difference.  But  it  seems  likely  that 
at  least  two  tyres  of  alkali  extractive  materials  were  present, 
that  probability  being  further  strengthened  by  the  fact  that 
the  sodium  carbonate  removed  the  red  color  from  the  benzene 
solution,  leaving  it  a strong  yellow,  even  though  the  sodium 
carbonate  fraction  was  much  smaller  than  the  sodium  hydroxide 
fraction . 


23. 


It  was  found  "by  experiment  that  a very  small  amount  of 
emodin  is  sufficient  to  give  a distinct  red  color  to  benzene. 
According  to  Beal  and  Obey  (24)  emodin  is  soluble  in  dilute 
sodium  carbonate;  chrysophanic  acid  is  insoluble  in  sodium 
carbonate,  but  soluble  in  dilute  sodium  hydroxide.  Hence  it  is 
probable  that  the  emodin  was  removed  by  the  sodium  carbonate, 
and  that  the  remaining  material  contained  chrysouhanic  acid, 
which  is  yellow.  Unfortunately  the  latter  fraction  was  over- 
heated or  oxidized  in  evaporating  the  solvent,  and  identifica- 
tion was  made  impossible. 

While  the  resinous  material  which  was  hydrolyzed  probably 
contained  frangulin,  the  aqueous  solution  could  not  oe  shown  to 
contain  rhamnose,  or  for  that  matter  any  reducing  sugar,  or 
even  a carbohydrate.  The  solution  contained  sufficient  alcohol 
for  preservative  and  was  kept  in  a stoppered  flasx  until  exam- 
ined. Nevertheless  this  was  the  only  case  in  which  emodin  was 
obtained  in  sufficiently  pure  state  to  permit  crystallization. 

It  is  possible,  during  the  concentration  of  the  hydrolyzed 
solution  for  precipi tation  in  water,  that  the  small  amount  of 
hydrochloric  a,cid  present  was  sufficient  to  convert  the  mannose 
to  methyl  furfural,  which  would  be  lost  by  volatilization. 

The  oil  obtained  from  the  seeds  has  promising  possibilities. 
As  shown  by  its  constants  it  resembles  sunflower,  maize,  and 

(Sherman)  (25). 


cottonseed  oils. 


. 


J 


r, 


24. 


Rh 

pi 

.Prang . 
eed  Oil 

Sunflower 
Seed  Oil 

Maize  Oil 

Cottonseed 

Oil 

Saponif i cation 

No.  - - - - - 

193.8 

188  - 196 

188  - 194 

190  - 197 

Iodine  No.  - - 

118.9 

104  - 135 

111  - 124 

104  - 116 

Spec.  Gravity 
15.5°  C.  

0.9183 

0.920-0.927 

0.921-0.926 

0 . 920—0 . 925 

Index  of  refr. 
15.5°  G.  

1.4749 

1.474-1.473 

1.475-1.47 7 

1.473-1.476 

It  seems  reasonable  to  exoect  that  the  oil  expressed  or 
extracted  simply  from  the  ground  dry  seeds,  and  containing  its 
normal  content  of  the  emodin,  etc.,  would  make  an  excellent 
lubricant  and  cathartic. 

Summary . 

The  analysis  of  the  fruit  of  Ehamnus  Prangula  indicates 
emodin  and  possibly  chrysoohanic  acid,  glucose,  a pentose,  a 
small  amount  of  light  brown  gum,  tannic  acid,  malic  acid, 
succinic  acid,  a resin  (which  was  probably  frangulin,  though 
no  rhamnose  was  found),  a deep  purplish-red  coloring  matter 
soluble  in  alcohol,  and  a fatty  oil  constituting  approximately 
eighteen  Per  cent  of  the  weight  of  the  dry  seeds. 


25 


Bibliography . 

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Rhamnus  Barbs.  Jour.  Am.  Pharm.  Assn.  V.  4 nt.  1,  p.65. 

(2) .  Matthioli , Petri  Andreae,  1548:  Gommentarii  in  VI  libros 

Pedacii  Dioscorides  (Caspar  Bauhin  edition,  1593), 
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(3) .  Linnaeus,  Carl,  1753:  Species  Plantarum,  Thomae  edition 

(3d,  1764),  pp.  279,  280,  and  1671. 

(4) .  Gerber,  G.  ?.,  1823:  Analysis  of  the  Cortex  of  Rhamnus 

Frangula.  Brande's  Archiv.  der  Pharmacie,  vol.  26,  p.l. 

(5) .  Hubert,  1830:  About  the  Nature  of  the  Expressed  Juice  of 

the  Fruit  of  Rhamnus  Catharticus.  Brande's  Archiv. 
der  Phar.,  vol.  34,  p.  142. 

(6) .  Fleury,  1842:  About  Rhamnin.  Jour,  fur  Prakt.  Chemie, 

vol.  26,  p.  226. 

(7) .  Winkler,  F.  L.,  1849:  On  the  Bitter  Matter  (Cathartin) 

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Frangula  and  Rhamnus  Cathartica.  Buchner's  Repert. 
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(9) .  Buchner,  L.  A.,  1853:  A Hew  Yellow  Coloring  Material  in 

Frangula  Root  Bark.  Neues  Repert.  fur  die  Pharm., 
vol.  2,  p.  145. 

(10) .  Casselmann,  Arthur,  1357:  About  Frangulin.  Liebig’s 

Ann.  der  Chemie  und  Pharm.,  vol.  104,  p.  77. 

(11) .  Phipson,  L.  T. , 1853:  On  a Coloring  Matter  Obtained  from 

Rhamnus  Frangula.  Pharm.  Jour,  and  Trans.,  vol.  13, 

■n  OQ  cr 

U • ) ♦ 

(12) .  Lubly,  M.,  1866:  The  Chemical  Constituents  of  the  bark 

of  Rhamnus  Frangula.  Neues  Repert.  fur  Pharm.,  1866, 
p.  295. 

(13) .  Faust,  August,  1869:  Frangulin  and  its  decomposi tion 

Products.  Pharm.  Centralhalle , vol.  10,  p.  193. 

(14) .  Liebermann,  C.,  and  Waldstein,  M.,  1376:  Emodin  from 

Rhamnus  Frangula  Bark.  Ber.  der  Deutsch  Chem.  Ges., 
vol.  IX,  p.  1775. 


26. 


(15)  . 

(16)  . 

(17)  . 

(18)  . 

(19)  . 

(20) . 

(21). 
(22)  . 

(23)  . 

(24)  . 

( 25 ) . 

(26) . 

(27)  . 

(28)  . 


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